Substances Having Body Mass Redistribution Properties

ABSTRACT

There is provided a method for altering the distribution of body mass by altering the distribution of body mass by decreasing overall percentage fat and/or increasing the proportion of lean mass to fat mass comprising administering to a subject one or more compounds having the ability to alter body mass composition and/or ACE inhibiting activity or a physiologically acceptable derivative or prodrug thereof.

FIELD OF THE INVENTION

The invention relates to therapeutic formulations and methods foraltering body mass distribution. More specifically the invention relatesto therapeutic formulations comprising compounds, such as flavonoids,polyphenols, polypeptides, leucine and other branched chain amino acidsand dairy bioactives, for use in methods for altering body massdistribution.

BACKGROUND OF THE INVENTION

In this specification where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge; or known to be relevant to anattempt to solve any problem with which this specification is concerned.

Increasing Lean Body Mass

There are a number of diseases which involve cachexia (weakness andwasting of the body) as a symptom in which the body loses significantamounts of lean body mass. Examples of such diseases include diabetes,cancer, Alzheimers, bulimia nervosa and anorexia.

There is thus a need for a treatment which enables the body to increaseits percentage of lean body mass with a minimal increase, or ideally adecrease, in the percentage of fat mass.

Polyphenols

Polyphenols (compounds with two or more phenolic hydroxy groups) are aclass of phytochemicals found in a variety of sources including wine,grapes, cocoa and sugar cane. Polyphenols (or phenolics) all have acommon basic chemical component, that is, a phenolic ring structure.There are at least 8000 identified polyphenols in a number ofsubcategories, such as anthocyanins and catechins. Natural polyphenolscan range from simple molecules such as phenolic acid to large highlypolymerized compounds such as tannins. Conjugated forms of polyphenolsare the most common, where various sugar molecules, organic acids andlipids (fats) are linked with the phenolic ring structure. Differencesin this conjugated chemical structure account for different chemicalclassifications and variation in the modes of action and healthproperties of the various compounds.

Polyphenols are considered to have a number of health benefitsincluding:

antioxidant activity;

cancer preventative properties;

heart disease and hypertension protection;

antibiotic/antiviral activity;

anti-inflammatory activity;

ophthamological properties; and

protecting and strengthening blood vessels.

Polyphenols are responsible for the brightly colored pigments of manyfruits, vegetables and flowers (ranging from pink through scarlet,purple and blue), they protect plants from diseases and ultravioletlight and help prevent damage to seeds until they germinate.

Unfortunately, although the epidemiologic data for regular fruit andvegetable intake and disease prevention is strong, dietary supplementscontaining isolated phenolic antioxidants have not been extensivelystudied in terms of disease prevention. Products such as green tea, HCA(hyroxycitric acid) and inulin claim weight loss benefits based on theassumption that these products delay glucose absorption and/or regulateinsulin to control appetite. This has yet to be proven in controlledclinical trials with humans (Functional Food Update 01, National Centreof Excellence in Functional Foods, Australia. June 2006).

Sugar Cane

Anthocyanins are polyphenolic flavylium salts with sugar units attachedto the molecule and are derived chiefly from six anthocyanidins:pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvidin.These compounds differ in the position and number of hydroxyl groups inring B but all have a sugar unit at the 3 position and are watersoluble. With the exception of the petunidin group, representatives ofall other anthocyanin classes have been located in sugar cane.

The basic structure common to all anthocyanins is as follows:

Tea

Second only to water, tea is one of the most widely consumed beveragesin the world. Approximately 3.0 million metric tons of dried tea isproduced annually, 20% of which is green tea, 2% is oolong and theremainder is black tea (International tea committee Annual bulletin ofstatistics 2002). Black, oolong and green tea are produced from theleaves of the tea plant Caimellia sinensis, a member of the Theaceaefamily. Different varieties of tea are produced by varying the degree ofleaf oxidation. Green tea is produced by steaming freshly harvestedleaves at high temperatures, inactivating oxidative enzymes. Thispreserves the high polyphenol content found in green tea. Black tealeaves are the most oxidated, while oxidation of oolong tea leaves ismidway between green tea and black tea.

The majority of polyphenols in tea are flavonols, specificallycatechins. These small molecules react with one another during theoxidation process that produces black and oolong teas to form larger,highly colorful compounds called theaflavins and thearubigins.

There has recently been a lot of research into potential pharmaceuticalbenefits of the polyphenols extracted from tea. The most potentchemopreventive agent commonly extracted from tea is(−)-epigallocatechin-3-gallate (EGCG). There are also claims that greentea polyphenols can assist with weight loss because of its ability toincrease metabolism and fat burning noted whilst studying the effect ofpolyphenols on cholesterol levels in the blood. Medicines made from teapolyphenols have become part of the treatment for nephritis, chronichepatitis, and leukemia in China. In other countries, green teasupplements are available.

The basic structure common to all catechins is as follows:

Cocoa

Theobroma cocoa is a rich source of flavonoids including polyphenols.One study on the consumption of dark chocolate by humans has shown thatflavonoid rich chocolate improves endothelial function and increasesplasma epicatechin concentrations. However, that study found no changein oxidative stress measures, lipid profiles, blood pressure, bodyweight or body mass index [Engler et al, “Flavonoid-rich dark chocolateimproves endothelial function and increases plasma epicatechinconcentrations in healthy adults” J Am Coll Nutr 2004; 23(3):197-204].

Another study on the consumption of dark chocolate found no changes inthe total antioxidant capacity of plasma or in the oxidationsusceptibility of serum lipids. The study did find that cocoapolyphenols may increase the concentration of HDL cholesterol whereaschocolate fatty acids may modify the fatty acid composition of LDL andmake it more resistant to oxidative damage [Mursu et al “Dark chocolateconsumption increases HDL cholesterol concentration and chocolate fattyacids may inhibit lipid peroxidation in healthy humans” Free Radic BiolMed 2004 Nov. 1; 37(9):1351-9].

ACE Inhibitors

ACE is an important part of the renin-angiotensin-aldosterone system,one of the major endocrine systems in the body. ACE cleaves angiotensinI (ANG-I) to the potent vasoconstrictor angiotensin II (ANG-II) whichregulates major physiological functions of the body including bloodpressure, body sodium and fluid homeostasis which mediates its functionvia cellular receptors AT-1 and AT-2. ACE inhibitors have beendemonstrated to be useful in lowering blood pressure and in thetreatment of left ventricular dysfunction and diabetic neuropathy.

There have been a number of studies into the various roles of ANG-II:

organogenesis (Oliverio M I, Madsen K, Best C F, I to M, Maeda N,Smithies O, Coffinan T M. “Renal growth and development in mice lackingAT1A receptors for angiotensin II”. Am. J. Physiol. 1998;274:F43-F50);

formation of pre-adipocytes;

human preadipocytes express a high affinity for AT-1 receptor substypes(Crandall D L, Armellino D C, Busler D E, McHendry-Rinde B, Kral J G.“Angiotensin II receptors in human preadipocytes: role in cell cycleregulation”. Endocrinology 1999;140:154-158);

white adipose tissue has been reported to be an important site ofangiotensinogen production (Cassis L A, Saye J, Peach M J. “Location anddevelopment of rat angiotensin messenger RNA”. 1988; Hypertension11:591-596);

stimulate adipogenesis or formation of adipose (fat) cells (Darimont C,Vassaux G, Alihaud G, Negrel R. “Differentiation of preadipose cells:paracrine role of prostacyclin upon stimulation of adipose cells byangiotensin-II”. Endocrinology 1994;135:2030-2036; Saint-Marc P. Kozak LP, Ailhaud G, Darimont C, Negrel R. “Angiotensin-II as a trophic factorof white adipose tissue: stimulation of adipose cell formation”.Endocrinology 2001;142:487-492);

increase lipogenesis and triglyceride accumulation in preadipose cellsand human adipocytes (Jones B H, Standridge M K, Moustaid N.“Angiotensin-II increases lipogenesis in 3T3-L1 and human adiposecells”. Endocrinology 1997;138:1512-1519);

rats treated with an ACE inhibitor (losartan) exhibit a reduction inadipocyte size (Zorad S, Fickova M, Zelezna B, Macho L, Kral J G. “Therole of angiotensin-II and its receptors in regulation of adipose tissuemetabolism and cellularity”. Gen. Physiol. Biophys. 1995;14:383-391)

Collectively, these studies indicate that ANG-II plays an important rolein the development of adipose tissue.

Studies have also shown that ACE inhibitors may be useful in reducingweight gain.

In angiotensinogen deficient mice, weight gain is lower than in normalwild type mice despite food intake being similar for both genotypes(Massiera F, Seydoux J, Geloen A, Quignard_Boulange A, Turban S,Saint-Marc P, Fukamizu A, Negrel R, Ailhaud G. and Teboul M.“Angiotensinogen-Deficient mice exhibit impairment of diet-inducedweight gain with alteration in adipose tissue development and increasein locomotor activity”. Endocrinology 2001;142(12):5220-5225).

Overfeeding of rodents leads to increased production of ANG-II andchronic ANG-II infusion results in a dose dependant reduction in bodyweight (Cassis L A, Marshall D E, Fettinger M J, Rosenbluth B, Lodder RA. “Mechanisms contributing to angiotensin II regulation of bodyweight”. Am. J. Physiol. Endocrinol. Metab. 1998;274:E867-E876).

In obese human hypertensive patients, ANG-II increases in adipocytes andmay be a contributing factor in the development of insulin resistance.This may be aggravated by the inhibition of preadipocyte recruitment,which results in redistribution of fat to the liver and skeletal muscle.For this reason, ACE-inhibition may have potential in slowing thedevelopment of type 2 diabetes and pathophysiological roles of theadipose-tissue renin-angiotensin-receptor system in metabolic syndrome(Engeli S, Schling P, Gorzelniak K, Boschmann M, Janke J, Ailhaud G,Teboul M, Massiera F, Sharma A M. “The adipose-tissuerenin-angiotensin-aldosterone system: role in metabolic syndrome”. TheInternational Journal of Biochemistry & Cell Biology 2003;35:807-825.)

However, none of these studies disclose a method for changing body masscomposition, eg, a reduced fat mass and increased lean muscle mass.Increasing lean body mass is not necessarily associated with a weightloss. There is thus still a need for such a method to assist subjectssuffering from cachexia.

Dairy Bioactives, Leucine, ACE Inhibitory Peptides and Other BranchedChain Amino Acids

Milk bioactives, leucine and other branched chain amino acids arenatural angiotensin converting enzyme (ACE) inhibitors. ACE inhibitorypeptides may be released by proteolysis of milk proteins by lactic acidbacterial during cheese ripening. They may also be isolated from milkand whey during fermentation (Fitzgerald R J, Murray B A. “BioactivePeptides and lactic fermentations”. International Journal of DairyTechnology 2006;59(2):118-125). ACE inhibitory dairy peptides have anIC₅₀ values >520 μm and sufficient amounts may be delivered viafermented milks and extracts of fermented dairy products. Althoughweight reduction has been proposed using dairy products (Zemel M B etal. “Dairy augmentation of total and central fat loss in obesesubjects”. Int. J. Obes. Relat. Metab. Disord. 2005;29(4):391-7), therole in weight management has recently been questioned (Gunther C W etal. “Dairy products do not lead to alterations in body weight or fatmass in young women in a 1-y intervention”. Am. J. Clin. Nutr.2005;81:751-756).

Obesity

A method for increasing the proportion of lean body mass could also beuseful for treating subjects suffering obesity.

Every person has and needs fat tissue in their body. When there is toomuch body fat, the result is obesity. There are over 300 million obeseadults, according to the World Health Organization and 1.1 billionoverweight people worldwide.

The number of overweight and obese Americans has continued to increasesince 1960, a trend that is not slowing down. More than half of USadults are overweight (64.5 percent) and nearly one-third (30.5 percent)are obese. Each year, obesity causes at least 300,000 excess deaths inthe US, the and healthcare costs of American adults with obesity amountto approximately $100 billion. It is the second leading cause ofpreventable death after smoking.

Obesity increases one's risk of developing conditions such as high bloodpressure, diabetes (type 2), heart disease, stroke, gallbladder diseaseand cancer of the breast, prostate and colon. The tendency towardobesity is fostered by our environment: lack of physical activitycombined with high-calorie, low-cost foods. If maintained, even weightlosses as small as 10 percent of body weight can improve one's health.

Being obese and being overweight are not the same condition. Yourbathroom scale may give you a measure of your weight and help you followchanges in your weight, but it is not the best way to determine if youare overweight or obese, or at risk for developing obesity and itsrelated health conditions.

In order to determine whether a person is obese, both body mass index(BMI) and waist circumference is needed. You can have a BMI thatindicates you have a healthy weight, but still have a waist measurementabove the healthy range.

BMI: is a number based on both height and weight. It can help todetermine the degree to which a person may be overweight and gives areasonable assessment of total body fat for the general population. BMIcorrelates better with health conditions like heart disease and type 2diabetes than does weight itself. BMI is not perfect. Some people, likeathletes, may measure a high BMI but have more muscle than fat. BMI“cutpoints” are numbers used to help you determine if you are at ahealthy weight, overweight, obese or severely obese. It is important tonote that BMI is different to Health/Weight tables.

18.5 to 24.9=Healthy Weight

25 to 29.9=Over-weight

30 to 34.9=Obesity (class 1)

35 to 39.9=Obesity (class 2)

40 or more=Severe Obesity (class 3)

Waist circumference measurement is used to determine health risksrelated specifically to abdominal fat.

For Men: 40 inches or more

For Women: 35 inches or more

If your waist measurement is more than that listed above, and your BMIis between 25 and 34.9, you have an increased risk of developing type 2diabetes, hypertension and cardiovascular disease.

Causes of Obesity

There are many factors that contribute to causing obesity includinggenetics, the environment and behaviour.

Genes: Some individuals have a genetic tendency to gain weight and storefat. Although not everyone with this tendency will become obese, somepersons without genetic predisposition do become obese. Several geneshave been identified as contributors to obesity, and researchers areconstructing a Human Obesity Gene Map to identify genetic targets inhumans that may lead to the development of new treatments.

Environment: An environment that promotes healthy weight is one thatencourages consumption of nutritious foods in reasonable portions andregular physical activity. A healthy environment is important for allindividuals to prevent and treat obesity and maintain weight loss.Identifying and consciously avoiding high-risk situations in theenvironment can assist in weight control efforts.

Behaviour: Adopting healthy habits for lifelong weight control includeregular physical activity and nutritious eating. Specific behaviouralstrategies for weight loss and maintenance include: logging and trackingdiet and exercise patterns in a diary, eating a low calorie diet,limiting the amount of calories from fat, expending calories routinelythrough exercise, monitoring weight regularly, setting realistic goals,and developing a social support network.

The number of obese people in the world is increasing despite the aboveknowledge. There is thus a need for methods to modify body massdistribution.

SUMMARY OF THE INVENTION

It has surprisingly been found that some compounds will alter the body'sfood processing to such an extent that the overall body massdistribution is altered. In particular, the addition of these compoundsto food results in an increased proportion of lean mass to fat mass whencompared to the consumption of the same food without the addition ofthese compounds. In other words, these compounds can reduce the amountof fat which is produced from consumed food. These body mass alteringcompounds include polyphenols and milk bioactives.

It has also been found that flavonoids and polyphenols may have ACEinhibiting activity Without wishing to be bound by theory, it is thoughtthat the ACE inhibiting activity is related to the ability of thesecompounds to alter body mass composition. However, it is acknowledgedthat the ability of these compounds to alter body mass composition mayalso be related to antioxidant properties (ie polyphenols) and/orcalcium influx effects (ie milk proteins).

According to a first aspect of the invention there is provided a methodfor altering the distribution of body mass by decreasing overallpercentage fat and/or increasing the proportion of lean mass to fat masscomprising administering to a subject an effective amount of one or morecompounds having at least one hydroxyl group and the ability to alterbody mass composition or a physiologically acceptable derivative orprodrug thereof.

The first aspect of the invention also provides a method comprisingadministering to a subject a therapeutic formulation comprising aneffective amount of one or more compounds having at least one hydroxylgroup and the ability to alter body mass composition or aphysiologically acceptable derivative or prodrug thereof and anacceptable carrier.

The first aspect of the invention also provides a therapeuticformulation when used to alter the distribution of body mass bydecreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of one or morecompounds having at least one hydroxyl group and the ability to alterbody mass composition or a physiologically acceptable analogue,derivative or prodrug thereof and an acceptable carrier.

The first aspect of the invention also provides for the use of aneffective amount of one or more compounds having at least one hydroxylgroup and the ability to alter body mass composition or aphysiologically acceptable analogue, derivative or prodrug thereoftogether with a suitable carrier in the manufacture of a medicament foraltering the distribution of body mass by decreasing overall percentagefat and/or increasing the proportion of lean mass to fat mass.

In this specification, the term “compounds having at least one hydroxylgroup and the ability to alter body mass composition” refers to anycompound that contains a hydroxyl group which alters body masscomposition by decreasing percentage fat and/or increasing theproportion of lean mass to fat mass. The compounds may be sourcednaturally from animals or plants or be manufactured synthetically. Anexample of an animal source is snake venom which contains peptides.Examples of plant sources are polyphenols from green tea, wine, cocoa,sugar cane, sugar beet, sugar cane and sugar beet waste products,molasses and Chinese herbs such as Magnolia liliflora and Magnoliaofficinalis. Other examples of such compounds include (i) flavonoidssuch as anthocyanins, catechins, polyphenols, chalcones, flavonols,flavones and (ii) polypeptides, leucine and other branched chain aminoacids and dairy bioactives such as extracts of whey. Preferably, thecompound having at least one hydroxyl group and the ability to alterbody mass composition is selected from the group consisting offlavonoids, polyphenols, milk proteins, ACE inhibitory peptides,molasses, molasses extracts, high phenolic sugars and mixtures thereof.

According to a second aspect of the invention there is provided a methodfor altering the distribution of body mass by decreasing overallpercentage fat and/or increasing the proportion of lean mass to fat masscomprising administering to a subject an effective amount of one or morecompounds having ACE inhibiting activity or a physiologically acceptablederivative or prodrug thereof.

The second aspect of the invention also provides a method comprisesadministering to a subject a therapeutic formulation comprising aneffective amount of one or more compounds having ACE inhibiting activityor a physiologically acceptable derivative or prodrug thereof and anacceptable carrier.

The second aspect of the invention also provides a therapeuticformulation when used to alter the distribution of body mass bydecreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of one or morecompounds having ACE inhibiting activity or a physiologically acceptableanalogue, derivative or prodrug thereof and an acceptable carrier.

The second aspect of the invention also provides for the use of aneffective amount of one or more compounds having ACE inhibiting activityor a physiologically acceptable analogue, derivative or prodrug thereoftogether with a suitable carrier in the manufacture of a medicament foraltering the distribution of body mass by decreasing overall percentagefat and/or increasing the proportion of lean mass to fat mass.

In this specification, the term “compounds having ACE inhibitingactivity” refers to any compounds having ACE inhibiting properties andthe ability to alter body mass composition by decreasing percentage fatand/or increasing the proportion of lean mass to fat mass . Thecompounds may be sourced naturally from animals or plants or bemanufactured synthetically. An example of an animal source is snakevenom which contains peptides. Examples of plant sources are polyphenolsfrom cocoa, sugar cane, sugar beet, sugar cane and sugar beet wasteproducts, molasses, grapes, wine, fruit (berries, drupes, pomes,tropical fruits, juices), vegetables (bulbs, roots, tubers, leaves,stems), herbs, spices, beans, pulses, grains (barley, buckwheat, corn,millets, oats, rice, rye, sorghum, wheat), nuts (almonds, betel nuts,cashews, hazelnuts, peanuts, pecans, walnuts), oilseeds, plant oils,tea, coffee, beer, cider, seeds, green tea, Chinese herbs such asMagnolia liliflora and Magnolia officinalis and mixtures thereof. Otherexamples of such compounds include (i) flavonoids such as anthocyanins,catechins, polyphenols, chalcones, flavonols, flavones and (ii)polypeptides, leucine and other branched chain amino acids and dairybioactives such as extracts of whey. Preferably, the compound having ACEinhibiting properties is selected from the group consisting offlavonoids, polyphenols, milk proteins, cocoa, cocoa products, cocoaextracts, grape extracts, molasses, molasses extracts, high phenolicsugar and mixtures thereof.

According to a third aspect of the invention there is provided a methodfor altering the distribution of body mass by decreasing overallpercentage fat and/or increasing the proportion of lean mass to fat masscomprising administering to a subject an effective amount of one or morepolyphenols or a physiologically acceptable derivative or prodrugthereof.

The third aspect of the invention also provides a method comprisingadministering to a subject a therapeutic formulation comprising aneffective amount of one or more polyphenols or a physiologicallyacceptable derivative or prodrug thereof and an acceptable carrier.

The third aspect of the invention also provides a therapeuticformulation when used to alter the distribution of body mass bydecreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of one or morepolyphenols or a physiologically acceptable analogue, derivative orprodrug thereof and an acceptable carrier.

The third aspect of the invention also provides for the use of aneffective amount of one or more polyphenols or a physiologicallyacceptable analogue, derivative or prodrug thereof together with asuitable carrier in the manufacture of a medicament for altering thedistribution of body mass by decreasing overall percentage fat and/orincreasing the proportion of lean mass to fat mass.

In this specification, the term “polyphenols” refers to any polyphenolssourced or derived from cocoa, sugar cane, sugar beet, sugar cane andsugar beet waste products, molasses, grapes, wine, fruit (berries,drupes, pomes, tropical fruits, juices), vegetables (bulbs, roots,tubers, leaves, stems), herbs, spices, beans, pulses, grains (barley,buckwheat, corn, millets, oats, rice, rye, sorghum, wheat), nuts(almonds, betel nuts, cashews, hazelnuts, peanuts, pecans, walnuts),oilseeds, plant oils, tea, coffee, beer, cider, seeds, green tea,Chinese herbs such as Magnolia liliflora and Magnolia officinalis andmixtures thereof. Preferably, the polyphenol is sourced from molasses,molasses extracts, high phenolic sugar and mixtures thereof. Preferably,the polyphenols have a high antioxidant activity.

According to a fourth aspect of the invention there is provided a methodfor altering the distribution of body mass by decreasing overallpercentage fat and/or increasing the proportion of lean mass to fat masscomprising administering to a subject an effective amount of molasses oran extract thereof.

The fourth aspect of the invention also provides a method comprisingadministering to a subject a therapeutic formulation comprising aneffective amount of molasses or an extract thereof and an acceptablecarrier.

The fourth aspect of the invention also provides a therapeuticformulation when used to alter the distribution of body mass bydecreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of molasses or anextract thereof and an acceptable carrier.

The fourth aspect of the invention also provides for the use of aneffective amount of molasses or an extract thereof together with asuitable carrier in the manufacture of a medicament for altering thedistribution of body mass by decreasing overall percentage fat and/orincreasing the proportion of lean mass to fat mass.

The term “effective amount” is used herein to refer to an amount whichis sufficient to alter the distribution of body mass by increasing leanmass or decreasing fat mass. The proportion of lean mass to fat mass isincreased when either the amount of lean mass of a subject increases orthe amount of fat mass of a subject decreases. Note that a change in theproportion of lean mass to fat mass does necessarily involve a change inoverall weight. An example of an effective amount for animals is 1 to 2%of the diet. Assuming that a human normally consumes 1000 g of food perday and the normal consumption of polyphenols is 1 g/day, the effectiveamount is likely to be in the range from 2 to 20 mg/day, more preferably2 to 10 g/day.

The ability of a compound to decrease percentage fat and/or increase theproportion of lean mass to fat mass can be tested using the miceexperiment discussed in the examples. If a statistically significantchange is obtained when compared to the control then the compound can beused in the invention. A typical result in the mice experiment is adecrease in percentage fat of 8 to 12% or an increase in the proportionof lean mass to fat mass of 4 to 7%. For humans suffering cachexia, anincrease in the proportion of lean mass to fat mass of at least 1 to 2%would be ideal.

The term “therapeutic formulation” is a broad term which includesenteral and parenteral pharmaceutical preparations, nutraceuticals,supplements, functional foods and herbal preparations. Examples ofsuitable formulations include tablets, powders, chewable tablets,capsules, oral suspensions, suspensions, emulsions or fluids, children'sformulations, enteral feeds, nutraceuticals, suppositories, nasalsprays, drinks and food products. The carrier may contain any suitableexcipients such as starch or polymeric binders, sweeteners, coloringagents, emulsifiers and coatings. Preferably, the carrier is a foodproduct or food ingredient such as sugar or chocolate.

The therapeutic formulation may be in any form appropriate foradministration to the subject. The therapeutic formulation may beadministered topically, orally or by any other route of administration.

The term “subject” as used herein refers to an animal. There is nolimitation on the type of animal that could benefit from the presentlydescribed formulations and methods. Preferably, the subject is a mammaland more preferably a human. An “animal” also includes livestock speciessuch as cattle, horses, sheep, pigs, goats, donkeys and poultry birdssuch as chickens, ducks, turkeys and geese or domestic animals such ascats and dogs. A subject, regardless of whether a human or non-humananimal, may also be referred to as an individual, animal, patient, hostor recipient. The formulations and methods of the present invention haveapplications in human medicine, the cosmetic and aesthetic industries,veterinary medicine as well as in general, domestic and wild animalhusbandry.

DRAWINGS

Various embodiments/aspects of the invention will now be described withreference to the following drawings in which (asterixes highlightsignificant differences):

FIG. 1 shows the extraction method used in Example 4.

FIG. 2 shows the Bone Mineral Content results from Example 6.

FIG. 3 shows the Lean Muscle Mass results from Example 6.

FIG. 4 shows the Fat Mass results from Example 6.

FIG. 5 shows the Percentage Fat results from Example 6.

FIG. 6 shows the Total Body Weight by DEXA results from Example 6.

FIG. 7 shows the Total Body Weight results from Example 6.

FIG. 8 shows the body weight at the time of glucose loading results forExample 8.

FIG. 9 shows the body weight at the time of DEXA analysis for Example 8.

FIG. 10 shows the percentage of fat mass at the time of DEXA analysisfor Example 8.

FIG. 11 shows the fat mass in grams at the time of DEXA analysis forExample 8.

FIG. 12 shows the lean mass in grams at the time of DEXA analysis forExample 8.

FIG. 13 shows the blood glucose results for Example 8.

FIG. 14 shows the food intake results for Example 8.

FIG. 15 shows the fluid intake results for Example 8.

FIG. 16 shows the liver fat oxidation results for Example 8.

FIG. 17 shows the body weight (A), proportion of body fat (B) andproportion of lean mass (C) in ACE +/+ (empty bars) and ACE −/− mice(filled bars). The values are mean±SEM (n=7 per group), *p<0.05;**p<0.01; ***p<0.001.

FIG. 18 shows the food (A) and water intake (B) in ACE +/+ (empty bars)and ACE −/− mice (filled bars). The values are mean±SEM (n=7 per group),***p<0.001.

FIG. 19 shows the proton density weighted axial MRI images across thebody of ACE +/+ (A) and for ACE −/− mice (B). Bright, white areas denotefat. Each series of images represents data from a single animal. Whitearrowhead indicates android fat.

FIG. 20 shows the rectal temperature (A) Spontaneous running wheelactivity (Distance run per day (B), speed (C) and proportion of fat inthe fecal matter (D) in ACE +/+ (Empty bars) and ACE −/− mice (filledbars). The values are mean±SEM (n=5 per group for rectal temperature,spontaneous running wheel activity measurements. ACE (−/−): n=6 and ACE(+/+): n=7 for fecal fat analysis).

EXAMPLES

Various embodiments of the invention will now be described withreference to the following non-limiting examples.

Example 1

This example compares the phenolic content and antioxidant activities ofphenolic powders which may be used in the methods of the invention.

Methods

The phenolic content and antioxidant activities of three phenolicpowders, the molasses phenolic powder produced at IFT (InternationalFood Technology Company), Hansen's Grape Extract HW 65-10 phenolicpowder, and the Vinlife™ grape seed extract powder, were compared. Thepowders were dissolved in 80% methanol at a concentration of 5 mg/ml.Further dilution with water was required to achieve concentrationsappropriate for the respective assays. The results of these assays areshown in Table 1 (below).

Results

The data in Table 1 allows the relative antioxidant efficiency of thepowders to be compared. Table 2 shows the specific activities of thethree powders, i.e. the number of antioxidant units per phenolic unit.

TABLE 1 Phenolic content and antioxidant activity of three phenolicpowders Phenolic Content Antioxidant Content (mg catechin (mg gallicacid Powder eqs/gram) eqs/gram) Molasses powder 254 32.2 Hansen's HW65-10 Grape 775 144 Extract Vinlife Grape Seed Extract 533 105

TABLE 2 Specific antioxidant activity of three phenolic powders SpecificActivity (gallic Powder acid eqs/catechin eqs) Molasses powder 0.127Hansen's HW 65-10 Grape Extract 0.188 Vinlife ™ Grape Seed Extract 0.197

Discussion

These results show that the molasses powder has a lower content ofphenolics than the other 2 powders and a lower specific antioxidantactivity. This is likely due to differences in the phenolic profilesbetween the various powders. HPLC analysis suggests that the molassespowder does not contain many of the simple phenolic acids, such asgallic acid, which are very powerful antioxidants. These compoundsappear to be insufficiently hydrophobic to bind to the XAD 16 resin.However, different extraction methods are likely to be able to extractsuch smaller hydrophilic compounds and they may be included into amolasses extract for use in a method according to the invention.

Example 2

This example investigates the antioxidant capacity in phenolic-fortifiedchocolate compared to non-fortified chocolate.

Method

The antioxidant capacity of 6 pieces of control milk chocolate (1 piecefrom each row of an approximately 100 g block) and 12 pieces ofphenolic-fortified milk chocolate (2 pieces from each row alternating1^(st) and 3^(rd), 2^(nd) and 4^(th)) were chosen for assay. The milkchocolate was provided by Cool Health Pty Ltd. A sample of each,weighing between 1.7 and 2 g, was weighed accurately and added to a 50ml tube. The chocolate samples were defatted by the addition of 20 mlheptane. The samples were centrifuged and the heptane decanted. Thesamples were left open in a fume hood to remove traces of heptane. Theantioxidants were extracted using 2×20 ml aliquots of 80% methanol, thefirst a 2 hour extraction and the second an overnight extraction. Theprimary and secondary extract were added together and assayed induplicate using the ABTS method after a 5-fold dilution in water.

Results

TABLE 3 Antioxidant capacity in chocolate Control chocolate Fortifiedchocolate Antioxidant Antioxidant Sample Capacity Capacity (Row, (mgcatechin Sample (mg catechin Position) equivs/g) (Row, Position)equivs/g) 1, 1 1.638 1, 2 1.832 2, 1 1.578 1, 4 1.857 3, 4 1.572 2, 12.022 4, 2 1.634 2, 3 1.859 5, 3 1.547 3, 4 1.924 6, 4 1.557 4, 1 1.9144, 3 1.937 5, 2 1.971 5, 4 1.936 6, 1 2.016 6, 3 1.900

Discussion

The antioxidant capacity of the control chocolate was 1.587±0.039 mgcatechin equivalents per gram (mean±standard deviation). The antioxidantcapacity of the phenolic-fortified chocolate was 1.961±0.142 mg catechinequivalents per gram. This represents an increase of 21.2% compared withthe control chocolate. It is thus possible for an effective amount ofpolyphenols to be added and uniformly distributed in a chocolate matrixto produce a formulation suitable for use in the methods according tothe invention.

Example 3

This example investigated the polyphenol content of extracts of varioussugar cane products at different stages in the sugar refining process. Acatechin equivalent assessment of first expressed juice, final juice,syrup, molasses, low pol sugar, mill mud, cane tops and foam wasundertaken.

Results

TABLE 4 Antioxidant potential of various sugar cane extracts TotalAntioxidant Potential (CE = catechin equivalents) Sample (mg CE/mL) (mgCE/g dry matter) First Expressed Juice 0.75 3.40 Final Juice 0.12 8.76Syrup extracted from 2.89 3.43 the clarified juice Molasses 23.58 30.00Low pol Sugar — 2.34 Filtrate 0.44 3.64 Cane tops 0.44 13.54 Foam 0.233.75 Mill Mud — 3.17 Raw Sugar 0.44 —

TABLE 5 Antioxidant potential of sugar cane extracts vs other polyphenolsources Polyphenols Anti-oxidants Sample (mg catechin equivs/g)(μmoles/g) Dark Chocolate 23.9 NT Milk Chocolate 7.25 18.3 Cocoa liquor41.8 110 Grape Seed Powder 301.5 1146 Grape Skin Extract 54.5 181 MixedBerry Snack 12.3 9.33 Mixed Juice 3.35 NT Mill mud 14.7 26.8 Molasses17.87 32.58 Raw sugar 0.25 0.44

The analysis revealed that the extracts from molasses and mill mudcontain a significant amount of polyphenols and thus could be added to aformulation suitable for use in the methods according to the invention.

Example 4

This example demonstrates the production of a sugar product containingpolyphenols which can be used in a formulation for use in a methodaccording to the invention. The flowchart in FIG. 1 illustrates theprocess used to produce a sugar cane molasses extract high inpolyphenols. The extraction of sugar cane molasses is discussed in moredetail in international patent application no 2005/117608.

A High Pol Sucrose base was prepared which comprised 99% total sucrose,glucose and fructose (wherein the amount of glucose and fructose was nomore than 0.5%) and 1% of a mixture of organic acids, minerals,polyphenols, antioxidants and polysaccharaides. This mixture consistedof the following:

600 to 2100 micrograms per gram of a mixture of trans-aconitic acid,oxalic, cis-aconitic, citric, phosphoric, gluconic, malic, succinic,lactic, formic and acetic acids, wherein most of the mixture consistedof trans-acotinic acid in an amount in the range from 200 to 600micrograms per gram;

150 to 600 micrograms per gram of minerals with the ratio of calcium tomagnesium to potassium being 50:15:35;

0.2 to 0.5 mg catechin equivalents per gram of polyphenols;

antioxidants so that the antioxidant activity is in the range of 0.4 to1.2 micromoles per gram; and

20 to 60 micrograms per gram of polysaccharides.

A sweetener high in phenolics was prepared by combining the High PolSucrose base with the extract high in polyphenols obtained above.

Electrospray Mass Spectrometry (ES/MS) was conducted on a MicromassPlatform ES/MS. The samples were dissolved in Methanol/Water (80:20) andinjected into a 20 μl loop and eluted with methanol/water (80:20) at 20μl/min. MS analysis was conducted in negative ion mode with a conevoltage of 40 kV and a mass range of 50-700 Da.

The sugar product contained a significant amount of polyphenols and thuscould be added to a formulation suitable for use in the methodsaccording to the invention.

Example 5

This example demonstrates the production of a commercial chocolateproduct containing polyphenols which can be used in the method accordingto the invention.

Infusion of Currants

Infusion Mixture: The following mixture (25 litres) was sufficient toinfuse 125 kg of currants which is sufficient for 1000 kg of chocolate.

20 litres Wine eg Shiraz, Merlot or Pinot Noir)

5 litres Grape Skin/Seed Extract

125 ml Flavouring

Mix the above well in a large vessel at room temperature Stir slowly toensure that the grape skin/seed extract and flavour is well blendedmixed with the wine.

The flavouring can be any natural or synthetic flavours depending uponthe specific note and profile desired in the finished chocolate. Theflavour may have an alcoholic, monosaccharide, polysaccharide,polydextrose, polydextrin, dextrin, polyol, starch, propylene glycol,vegetable oil, triglyceride or other suitable base/carrier.

A non-alcoholic infusion mix can also be utilised if required bysubstituting the wine variety by a non-alcoholic, de-alcoholisedvariety. In addition a range of non-alcoholic or de-alcoholised flavourscan also be added to the infusion mix to improve taste anddeliverability of the currants in the chocolate.

Infusing the Currants: Combine currants and infusion mixture in a vesselwhich can be rotated to fully mix the contents. Rotate the vesselregularly for the next 24 hours. Filter/strain off any excess liquid andspread the infused currants on a drying rack and place in a warm room(40° C.) with air flowing across the currants overnight.

Preparation of Chocolate Containing Grape Seed Powder and Flavouring

Base Chocolate Recipe (per 500 kg (0.5 t batch)):

Ingredient Amount Castor Sugar 200 kg Full cream milk powder 70 kg CocoaLiquor (Ivory Coast) 175 kg Cocoa Butter Deodorised 50 kg Soy Lecithin -(Add half initially and half 2.5 kg 30-60 mins before finishing conchingcycle) PGPR - (Add half, 1 kg, initially and 2.0 kg remainder afteraddition of flavours (to reduce viscosity).) Natural Vanilla Flavour -(Add 30 mins 2.0 kg before finishing conching cycle)

Add to conche in correct sequence and conche for 12-16 hours at 40° C.until average particle size of chocolate reaches less than 20μ (range18μ-20μ). The chocolate was then flavoured as one of shiraz, pinot ormerlot. The chocolate has a milk fat to cocoa butter ratio of 0.13.

The real varietal wine flavour in the chocolate can be enhanced byadding a range of flavours that not only enhance the flavour but serveto reduce the bitterness when higher than usual amounts of polyphenolsare added to promote health. A person skilled in the art of flavourchemistry will know which mix of flavours may be used to improvepalatability, mouth feel and other organoleptic properties.

Preparation Of Seed Powder (for 0.5 tonne batch of chocolate): Weigh out2.25 kg of Vinlife (Tarac Technologies) Grape Seed Powder and add to 5kg of melted (45° C.) cocoa butter. Add slowly with stirring and ensurethe powder is dispersed evenly throughout the cocoa butter. Avoidincorporation of air whilst mixing, but ensure that the powder is welldispersed in the cocoa butter.

Addition Of Seed Powder To Chocolate: To 0.5 tonne (500 kg) ofwine-flavoured chocolate held in holding tank at 40-45° C. add the 5 kgof cocoa butter containing the dispersed seed powder. Add slowly and mixin the tank for 5 minutes or until evenly dispersed.

Addition of Infused Currants to Chocolate

The filtered and drained currants (approx 5.5-5.8 kg) were mixed with 40kg of the flavoured and tempered chocolate. The mixture must be mixedwell to ensure an even distribution of the currants.

The currant/chocolate mixture is then moulded and cooled.

By using dried currents or fruit infused with wine and water-solublepolyphenols dispersed in cocoa butter, difficulties typicallyexperienced with addition to foods such as chocolate can be overcome.Taste can further be improved using wine flavours and a uniquelypalatable product can be produced with enhanced polyphenol content,antioxidant and ACE inhibitory activity for use in the methods accordingto the invention.

Example 6

In this example, sugar polyphenols or molasses extract from example 4were tested to determine the effect on body mass distribution of mice.

Method

In this experiment disease free six week old male C57B1/6J mice (n=65)were used. The mice were purchased from the Animal Resource Centre,Canning Vale, WA, Australia.

Several days after arrival in the animal house, mice were shifted fromtheir normal chow diet (3% fat) to a high fat-high carbohydrate diet(21% fat, 20% protein, 49% carbohydrate, 5% cellulose, 5% vitamins andminerals). The diets were specially formulated by Specialty Feeds, GlenForrest, Wash., Australia. All animals were housed 2 per group at 19-21°C. with a 12:12 light dark cycle.

Three groups of mice (n=13 mice per group) were maintained on the highfat-high carbohydrate diet containing (1) 1% polyphenol-containingpowder; (2) 2% polyphenol-containing powder; (3) molasses; (4) 1%sucrose (control). The diets used in this example were made by combining98-99% of the base diet plus 1-2% of the additives noted above. Theanimals were fed the diets for 9 weeks.

During the 9 week period, food and water intakes and body weight wasmeasured weekly.

At week 9 body composition of mice was determined using Dual EnergyX-ray Absorptiometry (DEXA).

Dual Energy X-ray Absorptiometiy (DEXA): Whole body composition of micewas assessed using DEXA (Norland XR-36) equipped with software packageoptimized for small animals. The mice were scanned under lightanaesthesia (Ketamil and Rompun). A whole body scanning mode was usedproviding information such as % body fat, bone mineral content (BMC),bone mineral density (BMD), and Lean mass. Animals were placed in theprone position at the centre and parallel to the long axis of the scantable.

Results and Discussion

Polyphenol powder and molasses added to the high fat diet, at both 1 and2% (PP1%, PP2%), decreased body fat (in grams—see FIG. 4 or as a % ofbody weight—see FIG. 5) and increased lean mass (see FIG. 3). Bodyweight and bone mineral content was not significantly altered (FIGS. 2and 6). DEXA was undertaken after 9 weeks of dietary intervention. DEXAbody weight (sum of lean, bone and fat) was highly correlated with bodyweight measured on a balance (r=0.98).

In FIGS. 2 to 7, statistical analysis by one way analysis of varianceand subsequent Fisher LSD post hoc test; vs control, *p<0.05; **p<0.01;***p<0.001.

There were no differences in food or water intake (not shown). Thecurrent figures do not show that molasses decreased body fat; i.e., Fat%, mean (SEM), control=36.9 (2.3), molasses=30.2 (1.7), PP1%=26.3 (1.6),PP2%=25.0 (2.8).

Conclusion

The results clearly demonstrate that the polyphenol powder changed bodycomposition by significantly increasing lean muscle mass andsignificantly decreasing percentage fat. The mean reduction of 11.9% fatand 6% increase in lean muscle mass would significantly improve theprognosis for those suffering from obesity, diabetes and cachexia.

Example 7

In this example, the molasses extract of example 4 and which was used inexample 6 was tested for antioxidant capacity (ORAC) and influence onthe activity of α-glucosidase and α-amylase.

Materials and methods

Sample Preparation: Samples were ground and approximately 50 mg wassolubilized in 5 mL of methanol. The samples were vortexed, sonicatedfor 30 minutes, and centrifuged for 5 minutes (1900 RCF). Thesupernatant was collected and taken to dryness. Samples werere-solubilized in methanol at 10 mg/ml.

Molasses powder samples were directly water-soluble. Molasses powder wassolubilized in phosphate buffer (pH 7.4) at a concentration of 1 mg/ml,prior to the ORAC assay. The molasses powder sample was also extractedas above, to provide comparative ORAC data. Molasses powder wassolubilized in water prior to the a-amylase and a-glucosidase assays.

Oxygen Radical Absorbance Capacity (ORAC) assay: The ORAC assay employedin this study measured the antioxidant scavenging activity in the testsample, against peroxyl radicals induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) at 37° C. Fluorescein was usedas the fluorescent probe. Hydrophilic ORAC values were determined forthe samples.

The extracts/samples were assayed using the ORAC procedure in serialdilution (×4) with AWA (acetone: water: acetic acid; 70:29.5:0.5), andin quadruplicate, starting with the concentration relevant to thesample, depending on the approximated antioxidant capacity from aninitial screen. A green tea extract was included as a positive control,and the extract was prepared as per the sample preparation.

Molasses powder sample was directly solubilized in phosphate buffer (pH7.4), and assayed, with the exception of AWA being substituted withphosphate buffer (pH 7.4). A methanolic green tea extract was includedas a positive control, and was also solubilized in phosphate buffer (pH7.4).

Trolox, a water soluble analogue of vitamin E, was used as a referencestandard. A trolox standard curve was established from trolox standardsprepared at 100, 50, 25, and 12.5 μM in AWA.

Briefly, 20 μL samples/standards/control/blank (AWA), 10 μL fluorescein(6.0×10⁻⁷ M), and 170 μL AAPH (20 mM) were added to each well.Immediately after loading, the plate was transferred to the plate readerpreset to 37° C., and the fluorescence was measured 35 times at oneminute intervals. The fluorescence readings were referenced to solventblank wells. The final ORAC values were calculated using a regressionequation between the Trolox concentration and the net area under thefluorescein decay curve, and were expressed as micromole Troloxequivalents (TE) per g of sample.

Glucose Metabolism Enzyme Inhibition Assays

α-Glucosidase: Molasses powder sample was solubilized in water prior touse in this assay. Fucoidan was included as a positive control, and wasalso solubilized in water.

Glucosidase enzyme was solubilized in acetate buffer (50 mM, pH 4.5) ata concentration of 0.7 mg/mL. This provided a final concentration of 0.2U/mL. To a 96-well plate, 50 μL of enzyme was added to each well. Acorresponding set of wells was also included, in which acetate bufferwas added instead of enzyme. Sample/controls were then added to thewells (5 μL), in triplicate, followed by the substrate4-Nitrophenyl-α-D-glucopyranoside (final concentration 2 mM). The platewas covered, shaken and then incubated at 37° C. for 30 minutes. Thereaction was stopped with the addition of 0.2 M Na₂CO₃ (100 μL/well).Absorbance was measured at 405 nm, using a Victor² plate reader.

The absorbance of the wells containing sample, substrate, and bufferwere subtracted from the corresponding wells containing the glucosidaseenzyme and the percent inhibition by the samples was calculated comparedto the solvent controls.

α-Amylase: Molasses powder sample was solubilized in water. Acarbose wasincluded as a positive control. Acarbose tablets were crushed andsolubilized in 50% aqueous ethanol (56 mg/mL). The solution wassonicated and centrifuged at 2000 RCF for 10 minutes. The supernatantwas collected and stored at 4° C.

An Enzchek Ultra Amylase assay kit was used to determine the influenceof sample 1 on α-amylase activity (Molecular Probes E33651). Briefly, a1× reaction buffer (supplied with the kit) was prepared by diluting thestock 1:10 with distilled water. One vial of lyophilised starchsubstrate (DQ™ starch from corn, BODIPY® FL conjugate) was prepared byadding 100 μL of 50 mM sodium acetate (pH 4.0) and then 900 μL of 1×reaction buffer, followed by 20-fold dilution with 1× reaction buffer.An amylase stock solution was prepared by solubilising 0.5 mg/mL ofporcine α-Amylase (Sigma A3176) in distilled water. The amylase stockwas then diluted with 1× reaction buffer to provide a concentration of125 U/ml.

The assay was performed using a 96-well plate format. 100 μL of amylaseenzyme solution was added to each well, followed by the samples andcontrols (5 μL/well). The substrate solution was then added (95 μL/well)and the fluorescence (excitation at 485 nm, emission at 530 nm) wasmeasured using a Victor plate reader.

Results and Discussion

The yield from each product is presented in Table 6.

TABLE 6 Yield of extract from each sample Sample Sample mass (mg)Extract mass (mg) Yield (%) Molasses 49.8 34.3 69 Powder Green tea 48.516.3 34

Antioxidant Capacity: The antioxidant capacities of the samples,prepared by making methanolic extracts, are presented in Table 7. Themolasses powder sample demonstrated the greatest antioxidant capacity,with an ORAC value of 4395 μmol TE/of sample when an extract wasgenerated or 5020 μmol TE/of sample when dissolved directly in buffer(Table 8). Both values were considerably higher than the correspondinggreen tea extract.

TABLE 7 Antioxidant capacity of molasses powder extracted with methanol,compared to a green tea methanol extract (values are mean ± standarderror of the mean). Sample no. ORAC value (μmol TE/g of sample) MolassesPowder 4395 ± 229  Green tea 1793 ± 93.5

TABLE 8 Antioxidant capacity of molasses powder solubilized directly inphosphate buffer (pH 7.4), compared to a green tea methanol extract(values are mean ± standard error of the mean) Sample no. ORAC value(μmol TE/g of sample) Molasses Powder 5020 ± 375 Green tea 1467 ± 90 

Glucose Metabolism Enzyme Inhibition Assays

Inhibition of α-Glucosidase: Molasses powder sample 1 inhibitedα-glucosidase to a limited extent, compared to the facoidan control(Table 9). The data from this assay was problematic, as the molassespowder sample exhibited a high background absorbance, which wassubtracted from the corresponding wells containing the glucosidaseenzyme. This possibly has caused an over-estimation of the inhibition inα-glucosidase activity, as inhibition is calculated compared to thesolvent control, which had a relatively low background absorbance.

TABLE 9 Inhibition (%) of α-glucosidase by sample 1, compared tofucoidan (values are mean ± SEM) Sample Concentration (μg/mL) %Inhibition IC₅₀ Molasses 600 88.9 ± 0.7 194 μg/mL Powder 300 65.7 ± 0.1150 38.4 ± 0.5 75 12.9 ± 0.9 37.5  1.5 ± 0.2 18.7 −3.8 ± 0.5 Fucoidan37.5 97.4 ± 5.9 14 μg/mL 18.8 73.5 ± 7.5 9.4 19.3 ± 2.7 4.7  5.7 ± 3.1

Inhibition of α-Amylase

Sample 1 did not inhibit α-amylase activity, compared to the control,acarbose (Table 10). An IC₅₀ was not able to be calculated from thisdata because Sample 1 did not inhibit α-amylase activity sufficiently.An IC₅₀ could possibly be calculated should the sample be tested at muchhigher concentrations, but the biological relevance of such aconcentration is questionable.

TABLE 10 Inhibition (%) of α-amylase by sample 1 compared to acarbose(values are mean ± SEM) Sample Concentration (μg/mL) % Inhibition IC₅₀Sample 1 1200 9.5 ± 1.7 — 600 −3.6 ± 0.9  300 −22.6 ± 1.6  150 −30.4 ±1.6  Acarbose 1000 95.2 ± 30.1 147 μg/mL 500 80.8 ± 10.2 250 61.5 ± 10.3125 46.4 ± 9.1 

Conclusion

This example clearly supports examples 1 and 4 by demonstrating byanother method for measuring antioxidant capacity that the molassesextract is a potent antioxidant. The relative potency of molasses powderis as follows;

grape seed extract>grape extract>molasses powder>green tea

Products such as green tea, HCA (hyroxycitric acid), and inulin claimweight loss benefits based on the hypothesis that consumption of theseproducts will delay glucose absorption and/or regulate insulin tocontrol appetite. Glucose absorption is controlled by glucosidase andamylase. Molasses extract has weak glucosidase activity therefore itappears that the changes in body composition must be by other mechanismsof action. It is more likely that the mechanism involves inhibition ofACE.

Example 8

This example investigates the effect of polyphenols extracted from teaon body mass distribution.

Method

Animals and treatments: Male Sprague Dawley rats (n=48) were purchasedfrom the Animal Resource Centre (Canning Vale, Wash.) at 3 weeks of age.Animals were allowed to acclimate for 1 week on Purina rat chow andwater. From 4 weeks of age, all animals were provided with asemi-synthetic, high fat diet (15% fat, Table 6) (Specialty Feeds, GlenForrest, Wash.) and administered one of four fluid treatments: GreenTea, Black Tea, Epigallocatechin Gallate (EGCG) or water. Tea and teaextracts were given as 100% of their fluid intake. Rats were maintainedon the high fat diet and tea treatment until week 29. Food and fluidintakes were measured daily and body weights recorded weekly.

TABLE 11 Composition of high fat diet. Ingredient % Composition Sucrose10.93 Casein 20.00 Soya Oil 1.86 Cocoa Butter 2.51 Ghee (Butter Fat)5.31 Tuna Oil 0.20 Olive Oil 4.23 Flaxseed Oil 0.91 Cellulose 5.00Starch 31.5 Dextrinised Starch 13.2 dI Methionine 0.30 AIN_93_G_TraceMinerals 0.14 Lime (Fine Calcium Carbonate) 1.31 Salt (Fine SodiumChloride) 0.26 Potassium Dihydrogen Phosphate 0.69 Potassium Sulphate0.16 Potassium Citrate 0.25 AIN_93_G_Vitamins 1.00 Choline Chloride 50%w/w 0.25

Green and Black tea: Green and Black tea bags (Dilmah natural green tea™and Dilmah black tea™) were purchased from a local retail outlet. Tentea bags (approximately 2 g tea leaves/bag) were steeped in 1 litre ofboiling tap water in a covered container for 3 minutes. Tea bags werethen expelled of excess tea and the tea preparations made up to a 2litre volume with cold tap water. This approximated to 1 tea bag per 200ml of water. Tea preparations were made fresh every second day.

Epigallocatechin Gallate: Epigallocatechin Gallate (EGCG (98%), SapphireBioscience, VIC) was dissolved in the drinking water and administered ata dose of 1 mg/kg/day. EGCG preparations were made fresh daily.

Glucose Tolerance Testing: Animals were fasted overnight with ad libitumaccess to fluid. The following morning, rats were restrained and tailswere immersed in local anaesthetic (Xylocaine) for 1 minute. A smallsegment was cut from the tip of the tail, and the tail massaged from thebase to the tip until a small amount of blood appeared. Blood sampleswere collected (hemocue microcuvettes) and fasted basal blood glucosesamples were taken (Hemocue Glucostat blood glucose analyser). An oralglucose load (40% glucose, bolus, 2 g/kg body weight) was then deliveredby gavage and blood glucose measured at 30-minute intervals for 2 hours.

Dual Energy X-ray Absorptiometry (DEXA): Body composition was determinedby dual energy x-ray absorptiometry using a Hologic QDR-4000/WAbsorptiometer. Animals were lightly anaesthetised (Nembutal, I.P., 40mg/kg) and placed supine on the scanning platform. Tails were taped inplace and a whole body scan was taken. Fat, lean and total mass wasmeasured, along with the percentage fat ratio and bone mineral content.Total mass as measured by DEXA was highly correlated with mass measuredby weighing the animal (r=0.99).

Statistical analysis: Results from the glucose tolerance testing werecompared using two-way analysis of variance (repeated measures) and aone way analysis of variance was used to compare DEXA and plasma insulinresults. Both analyses were followed by the LSD test. Significance wasreached when p<0.05. All results are presented as mean±SEM.

Results & Discussion

FIGS. 8 to 16 show the results obtained.

No change in blood glucose levels were observed as a result of theintervention.

The body weight for rats on all treatments was similar. The polyphenolsdid not alter the overall body weight.

At 11 and 18 weeks, the percentage of fat mass for the green tea andblack tea treatments was significantly lower than that for the watercontrol. At 18 weeks, the percentage of fat mass result for EGCG wasalso significantly different. At 18 weeks, the grams of fat mass wassignificantly lower than that for the water control. The polyphenolscaused less fat mass to be produced when on the same food diet as thewater control.

At 11 and 18 weeks, the grams of lean mass for the green tea and EGCGtreatments was significantly higher than that for the water control. Thepolyphenols caused increased lean mass to be produced. The difference inthe polyphenol content between green tea and black tea is likely to bethe reason for the fact that black tea did not significantly alter thelean mass when compared to the water control.

Example 9

In this example, evaluation of angiotensin converting enzyme knock-out(ACE −/−) mice was undertaken to determine if they develop a phenotypeof reduced fat mass.

Materials & Methods

Mice: Male and female heterozygous ACE knockout mice (+/−) were obtainedfrom the laboratory of Pierre Meneton, Insern, U367, Paris, France. Theywere maintained on a C57BL/6J background in the animal house.Heterozygous (ACE +/−) mice were bred to produce wild type (ACE +/+) andhomozygous ACE null offspring (ACE −/−). Real time polymerase chainreaction incorporating dual labelled-Taqman® probe technology (AppliedBiosystems, Foster City, Calif.) was used for genotyping of ACE (−/−)and ACE (+/+) offspring. Mice were housed in individual plastic cageswith sloping grill lids (Wiretainers, Melbourne, Australia). Food(Barastoc, Mouse Breeder cubes, Barastoc Stockfeeds, Australia) wasavailable ad libitum on the sloping section of the lid and there wasfree access to tap water. The mice were maintained on a 12 hourlight/dark cycle. Age matched male ACE (+/+) and ACE (−/−) mice pairsthat were 12 months old and had been maintained in the same housingconditions were selected for the study. The amount of food and waterconsumed was monitored daily for one week.

In vivo visualisation of distribution of adipose tissue by MagneticResonance Imaging (MRI) Technique: Regional body fat distribution wasvisualised by magnetic resonance imaging (MRI). Images were acquired ona Bruker BIOSPEC 47/30 scanner, equipped with a horizontal 4.7 T Oxfordmagnet. Proton density weighted axial images with the followingparameters:number of slices, 20; slice thickness, 1 mm; field of view(FOV) 6 cm; matrix, 256×256; repetition time (TR), 815 ms; echo time(TE), 17.9 ms were acquired. Mice were anaesthetised by placing them inan induction chamber with an exposure to an Isoflurane (AbbottAustraliasia Pty Ltd, Sydney, Australia) concentration of 5% v/v inmedical grade air and subsequent reduction to a concentration of 2%.

Body Composition Analysis by Dual Energy X-ray Absorptiometry (DEXAM):The evaluation of the whole body composition of ACE (−/−) and ACE (+/+)mice were performed using DEXA (Hologic QDR 4500, Hologic Inc. USA)equipped with software package (version 3.07) optimized for smallanimals. The animals were scanned while in a prone position under lightanaesthesia (0.02 ml/g body weight) with a mixture of ketamine (0.75 mlof 100 mg/ml Ketaplex, Apex Lab.) and xylazine (0.25 ml of 20 mg/mlRompun, Bayer).

Blood Analyses: At the end of experiment, mice were sacrificed bybleeding from heart under anaesthesia by intraperitoneal injection of aKetamine and Xylazine mixture described above. Blood was collected withheparin coated syringes and hematocrit was measured immediately afteraspiration of a sample to a capillary tube followed by centrifugation ina micro centrifuge (HERMLE Z 233 M-2, Medos Company Pty Ltd, Victoria,Australia.) for 5 minutes at 10,000 rpm. Subsequently, the plasma wasseparated by centrifugation at 3,000 rpm for 15 minutes in arefrigerated centrifuge (Sorval-RT7) and stored at −80 degrees celciusuntil the biochemical analysis were completed. The plasma triglycerides,total cholesterol and glucose levels were measured by spectrophotometryaccording to the procedures described in commercially available kits(Beckman-Coulter Inc., Fulerton, Calif., USA). In ACE (−/−) and ACE(+/+) mice (n=6), plasma leptin was measured as previously described.

Measurement of Core Body Temperature (rectal temperature): Temperaturewas measured by K-type thermocouples connected to a dual channel Fluke52 (John Fluke Manufacturing) electronic thermometer. To measure therectal temperature, a thermocouple (coated in silicon at the tip) wasinserted 2 cm into the anal sphincter of each mouse. The tip of thethermocouple and connecting wires were coated with 5% w/v lidocaine gel(Xylocaine, Astra Pharmaceuticals) as a local anaesthetic and lubricant.The temperature measurements were taken at the same time on fourconsecutive days and the average was taken of those four measurements.

Spontaneous Physical Activity on Running Wheel: Animals were allowed for14 days ad libitum access to running wheels equipped with a speedometer(Sigma Sport BC 700 calibrated for running wheel radius) fitted to theindividual plastic cage with grill lid. The distance run (km) and speed(km/h) were measured daily over 10 days. The mice were allowed freeaccess to food and water.

Analysisfor Faecal Fat Content: Faeces were collected from mice cagesover the period of one week and kept in the freezer (−20 degreesCelsius) until analysis. Lipids were extracted from 5 g of faeces usinga 2:1 chloroform:methanol solution. The total lipid content wasdetermined gravimetrically after extraction for 24 hours at roomtemperature. The dry weight of the faeces was determined on the lipidextracted residue. The total dry weight of the faeces was determined byadding the weight of fat content to the dry weight of faecal residue.

Statistical Analysis: All data is reported as mean+/−SEM. Thedifferences between the two groups were analysed by a student t-test(Statistica, Statsoft, USA).

Results

TABLE 12 Plasma composition and hematocrit of ACE+/+ and ACE−/− miceParameter ACE+/+ ACE−/− Triglyceride (mmol/l, n = 7)  0.85 ± 0.26 0.47 ±0.06 Cholesterol (mmol/l, n = 7)  1.68 ± 0.14 1.97 ± 0.11 Glucose(mmol/l, n = 7) 14.31 ± 1.72 10.81 ± 0.87  Hematocrit (%, n = 5) 40.5 ±0.9  27.5 ± 1.1*** The values are expressed as mean ± SEM; ***p < 0.001(ACE−/− vs. ACE+/+)

Body Weight, Body Fat, Food and Water Intakes: In comparison to ACE(+/+) mice, the ACE (−/−) mice weighed 14-16% less (p<0.01); (FIG. 17A)and had 50-55% less body fat (p<0.001; FIG. 17B). ACE (−/−) mice had asignificantly increased proportion of lean body mass compared with ACE(+/+) mice (FIG. 17C).

Food intake was similar (FIG. 18A), but water consumption of the ACE(−/−) mice was more than double that of ACE (+/+) mice (p<0.001; FIG.18B). The blood leptin level of ACE (−/−)m ice tended to be lower thanthat of ACE (+/+) mice (1.5+/−0.3 vs. 8.1+/−2.8 nmol/l: F(1,4) df=5.60,p<0.07, n=3 per group) and was correlated with body fat (r=0.85,p<0.05).

Bone—No significant differences were observed between ACE (−/−) and ACE(+/+) mice in either proportion of bone mineral content (2.2+/−0.06 vs.2.1+/−0.05, n=7 per group) or bone mineral density (0.076+/−0.002 vs.0.078+/−0.001 g/cm², n=7 per group).

Visualization of Regional Fat Masses by MRI: The bright white areas inproton density-weighted MRI images are fat. Visual comparison of seriesof axial images demonstrated that adipose tissue was markedly reduced inACE (−/−) compared to ACE (+/+) mice (FIG. 19). This effect was mostnoticeable in abdominal fat mass, as indicated by the arrow.

Core Body Temperature, Spontaneous Physical Activity Level and FatExcretion: No significant differences were observed between ACE (−/−)and ACE (+/+) mice in core body temperature (FIG. 20A), spontaneousactivity (average distance run, FIG. 20B; speed, FIG. 20C), orproportion of fat in the faecal matter (FIG. 20D).

Hematocrit and Plasma Composition: In Comparison to ACE (+/+) mice, theACE (−/−) mice had a lower hematocrit (p<0.001). No differences wereobserved in plasma glucose, triglyceride (TG) or total cholesterollevels (Table 12).

CONCLUSION

Given the same physiological changes occurred using ACE deficient animalmodels and various polyphenol sources (tea, molasses and molassesextracts), the results support the inference that the polyphenols areacting via an ACE inhibiting mechanism.

The word ‘comprising’ and forms of the word ‘comprising’ as used in thisdescription and in the claims does not limit the invention claimed toexclude any variants or additions.

Modifications and improvements to the invention will be readily apparentto those skilled in the art. Such modifications and improvements areintended to be within the scope of this invention.

1. A method for altering the distribution of body mass by decreasingoverall percentage fat and/or increasing the proportion of lean mass tofat mass comprising administering to a subject an effective amount ofone or more compounds having at least one hydroxyl group and the abilityto alter body mass composition or a physiologically acceptablederivative or prodrug thereof.
 2. The method according to claim 1wherein the compound having at least one hydroxyl group and the abilityto alter body mass composition is selected from the group consisting offlavonoids, polyphenols, milk proteins, molasses extracts, highflavonoid sugars and mixtures thereof.
 3. The method according to claim2 wherein the polyphenols are sourced from the group comprising wine,cocoa, sugar cane, sugar beet, molasses, molasses extracts, sugar caneand sugar beet waste products and mixtures thereof.
 4. The methodaccording to claim 1 wherein the effective amount is 1 to 2% by weightof total food consumed.
 5. The method according to claim 1 wherein theeffective amount for a human is 2 to 20 g/day.
 6. The method accordingto claim 1 wherein the increase in the proportion of lean mass to fatmass of a human suffering cachexia is at least 1 to 2%.
 7. (canceled) 8.(canceled)
 9. (canceled)
 10. A therapeutic formulation when used toalter the distribution of body mass by decreasing overall percentage fatand/or increasing the proportion of lean mass to fat mass comprising aneffective amount of one or more compounds having at least one hydroxylgroup and the ability to alter body mass composition or aphysiologically acceptable analogue, derivative or prodrug thereof andan acceptable carrier.
 11. The therapeutic formulation according toclaim 10 wherein the therapeutic formulation is a functional food. 12.The therapeutic formulation according to claim 11 wherein the functionalfood is selected from the group consisting of chocolate products, sugarproducts and mixtures thereof.
 13. (canceled)
 14. A method for alteringthe distribution of body mass by decreasing overall percentage fatand/or increasing the proportion of lean mass to fat mass comprisingadministering to a subject an effective amount of one or more compoundshaving ACE inhibiting activity or a physiologically acceptablederivative or prodrug thereof.
 15. The method according to claim 14wherein the compound having ACE inhibiting properties is selected fromthe group consisting of flavonoids, polyphenols, milk proteins, cocoa,cocoa products, cocoa extracts, grape extracts, molasses extracts, highflavonoid sugar and mixtures thereof.
 16. The method according to claim15 wherein the polyphenols are sourced from the group comprising wine,cocoa, sugar cane, sugar beet, sugar cane and sugar beet waste productsand mixtures thereof.
 17. The method according to claim 14 wherein theeffective amount is 1 to 2% by weight of total food consumed.
 18. Themethod according to claim 14 wherein the effective amount for a human is2 to 20 g/day.
 19. The method according to claim 14 wherein the increasein the proportion of lean mass to fat mass of a human suffering cachexiais at least 1 to 2%.
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. Atherapeutic formulation when used to alter the distribution of body massby decreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of one or morecompounds having ACE inhibiting activity or a physiologically acceptableanalogue, derivative or prodrug thereof and an acceptable carrier. 24.The therapeutic formulation according to claim 23 wherein thetherapeutic formulation is a functional food.
 25. The therapeuticformulation according to claim 24 wherein the functional food isselected from the group consisting of chocolate products, sugar productsand mixtures thereof.
 26. (canceled)
 27. A method for altering thedistribution of body mass by decreasing overall percentage fat and/orincreasing the proportion of lean mass to fat mass comprisingadministering to a subject an effective amount of one or morepolyphenols or a physiologically acceptable derivative or prodrugthereof.
 28. The method according to claim 27 wherein the polyphenols issourced or derived from cocoa, sugar cane, sugar beet, sugar cane andsugar beet waste products, molasses, grapes, wine, fruit, vegetables,herbs, spices, beans, pulses, grains, nuts, oilseeds, plant oils, tea,coffee, beer, cider, seeds, green tea and mixtures thereof.
 29. Themethod according to claim 27 wherein one or more of the polyphenols havea high antioxidant activity.
 30. The method according to claim 27wherein the effective amount is 1 to 2% by weight of total foodconsumed.
 31. The method according to claim 27 wherein the effectiveamount for a human is 2 to 20 g/day.
 32. (canceled)
 33. A therapeuticformulation when used to alter the distribution of body mass bydecreasing overall percentage fat and/or increasing the proportion oflean mass to fat mass comprising an effective amount of one or morepolyphenols or a physiologically acceptable analogue, derivative orprodrug thereof and an acceptable carrier.
 34. (canceled)
 35. A methodfor altering the distribution of body mass by decreasing overallpercentage fat and/or increasing the proportion of lean mass to fat masscomprising administering to a subject an effective amount of molasses oran extract thereof.
 36. The method according to claim 33 wherein themolasses or extract thereof has a high antioxidant activity.
 37. Themethod according to claim 33 wherein the effective amount is 1 to 2% byweight of total food consumed.
 38. The method according to claim 33wherein the effective amount for a human is 2 to 20 g/day.
 39. Themethod according to claim 33 wherein the increase in the proportion oflean mass to fat mass of a human suffering cachexia is at least 1 to 2%.40. (canceled)
 41. A therapeutic formulation when used to alter thedistribution of body mass by decreasing overall percentage fat and/orincreasing the proportion of lean mass to fat mass comprising aneffective amount of molasses or an extract thereof and an acceptablecarrier.
 42. (canceled)